P986: CYTOGENETIC DIAGNOSIS WITH NEXT-GENERATION CYTOGENETICS BY OPTICAL GENOME MAPPING IN PATIENTS WITH MYELOFIBROSIS.
Authors: Á. Díaz González, G. Avetisyán, E. Mora, M. Santiago, A. Liquori, S. Furió, S. García, A. Villalba, Á. López, J. V. Gil, C. García, E. González, C. Martínez, B. Fernández, M. Guaita, B. Martín, L. Cordon, E. Barragán, J. de la Rubia, J. Cervera, E. Such
Journal: HemaSphere (2022)

Background: The prognosis of myelofibrosis patients is highly variable. Due to this heterogeneity, it is important to have prognostic tools that correctly stratify the patients. The most current prognostic scales such as the MIPSS70+ v2.0 or the GIPSS require cytogenetic data. The main limitation in classifying these patients is the lack of information on cytogenetic alterations because of bone marrow fibrosis and absence of metaphases. Recently, a new strategy has emerged that characterize all cytogenetic alterations using Optical Genome Mapping considered Next Generation Cytogenetics which overcomes the limitations of conventional cytogenetics by using ultra high molecular weight DNA instead of metaphases. This DNA can be obtained from peripheral blood or bone marrow samples. Aims: The main objective of this project is to perform a complete genomic characterization of 10 patients with myelofibrosis by karyotype, FISH, Next Generation Sequencing (NGS) and Optical Genome Mapping (OGM). Methods: We have performed the karyotype staining the chromosomes by G-banding and the results were reported according to the International System for Human Cytogenetic Nomenclature (ISCN, 2020). FISH was performed to discard rearrangements of PDGFRA, PDGFRB, FGFR1 and JAK2. NGS libraries were prepared using a panel SOPHiA Myeloid Solution™ kit which includes SNV and CNV of 30 genes related to myeloproliferative neoplasms and leukemia. For study by OGM we obtained ultra-high molecular weight (UHMW) DNA from peripheral blood samples which are labeled with DLGreen fluorophores using Enzyme 1 (DLE-1) reactions following the manufacturer’s protocols (Bionano Genomics). Labeled DNA was loaded on a Saphyr chip and run on a Saphyr instrument (Bionano Genomics). The novo genome map assembly will be performed using BionanoSolve™ and structural variants will be called against the human reference hg38 assembly. Data was analyzed with Bionano Access™ (Bionano Genomics). Results: Preliminary results were obtained: - Patient 1 had unsuccessful karyotype and JAK2 (p.Val617Phe, VAF 13%) mutation by NGS. - Karyotype result of patient 2 suggest that is normal: 46,XY[6]. NGS detected pathogenic mutations in JAK2 (p.Val617Phe, VAF 41%), SRSF2 (p.Pro95His, VAF 42%) and ETV6 (p.Arg105*, VAF 41%) and likely pathogenic mutation in ASXL1 (p.?; c.1720-2A>G, VAF 45%). - Patient 3 had a normal karyotype: 46,XX[18] with CALR type 1 mutation (p.Leu367Thrfs*?, VAF 37%) by NGS. We have not detected rearrangements by FISH in none of the 3 patients. 1500 Gbp of DNA were collected to perform the OGM for each patient and we selected structural variants that were present in less than or equal to 0,5 % of the control samples with the same enzyme. Patients 1 and 2 who had unsuccessful karyotype could be characterized by OGM. We have found by OGM a median of 1 insertion (range 0-4), 8 deletions (4-10) and 2 duplications (0-3). No inversion or translocation were detected. No cytogenetic alterations classified as very high-risk karyotype in MIPSS70+ v2.0 scale were detected. After an exhaustive analysis of these structural alterations initially detected, all alterations were ruled out as they are present in general population and therefore correspond to a normal karyotype. Summary/Conclusion: Optical Genome Mapping suggest to be very promising diagnostic tool that can complete the study of patients with myelofibrosis, especially in those with non-assessable karyotype. The extension to a larger number of patients will confirm the results obtained and detect variables that remain cryptic to the karyotype.